Urapidil alleviates ovarian torsion detorsion injury via regulating oxidative stress, apoptosis, autophagia, and inflammation

Objective(s): This study aimed to determine anti-inflammatory, antioxidant, and antiapoptotic properties of urapidil (Ura) against ovarian torsion detorsion (T/D) injury in rats. Materials and Methods: 40 female Wistar albino rats were grouped as sham, T/D, T/D+dimethyl sulfoxide (DMSO), T/D+Urapidil (Ura) 0.5 mg/kg (low dose), and T/D+Urapidil (Ura) 5 mg/kg (high dose) groups. In treatment groups, Ura was administered intraperitoneally just before detorsion. Biochemical parameters (TAS, TOS, MDA, MPO, and SOD) and immunohistochemical (IL-1β, TNF-α, NF-κB, LC3B, and Caspase-3) analyzes were performed. Results: In the T/D group, OSI and MPO levels were elevated significantly while TAS values decreased compared with the sham group. A significant difference occurred in the low dose treatment group in TAS and OSI levels compared with the T/D group. In the high dose treatment group, significant elevation in TAS but reduction in OSI and MDA levels were observed compared with the T/D group. Immunohistochemical staining resulted in IL-1β, TNF-α, NF-κB, LC3B, and caspase-3 immunopositivity in the T/D group, while Ura treatment decreased those parameters. Intensive congestion and hemorrhage were observed in the T/D group, but contrary to this, treatment groups had alleviated congestion and hemorrhage. Conclusion: These results suggest that Ura demonstrated protective effects against ovarian T/D injury via anti-oxidative, anti-inflammatory, and anti-apoptotic features.


Introduction
Acute ovarian torsion is an emergency characterized by tissue damage. In the clinic, acute ovarian ischemia can be observed due to ovarian torsion, which ranks 5 th among gynecological emergencies (1). If there is severe necrosis, oophoropexy might be considered but laparoscopic detorsion is recommended to conserve the ovaries and fertility (2). The main cause of ovarian tissue damage in ovarian torsion is tissue ischemia and subsequent reperfusion injury due to formation of excess reactive oxygen molecules after detorsion, which can be defined as ischemia-reperfusion (I/R) injury (1,3). Following detorsion, regaining of the blood flow results in reactive oxygen species (ROS) formation. ROS affect biological molecules by clashing with their biological functionality (4,5).
ROS induce immune cytotoxic responses in various cell types (6). Oxidative stress stimulates phosphorylated protein activation, including protein kinases that play a key role in cell cycle regulation. Superoxide dismutase (SOD) and several members of the antioxidant system can suppress the cell cycle progression (7,8).
During torsion detorsion (T/D), overproduction of ROS primarily leads to tissue damage. Enzymatic and non-enzymatic cellular anti-oxidant mechanisms function synergistically. SOD is produced directly in the intracellular microenvironment and prevents tissues from ROS and oxidative damage. Malondialdehyde (MDA), the final lipid peroxidation product, is produced with ROS and is generally preferred as an indirect indicator of ROS (9). Excessive ROS production may lead to cellular damage resulting in apoptosis of the cell (10). The total antioxidant status (TAS) represents the antioxidative system while the total oxidant status (TOS) demonstrates the oxidative mechanisms (11). The vasodilator drug urapidil (Ura), the agent that increases tissue oxygen support and has antioxidant effects, was examined in previous studies (12,13). Ura includes metabolites with antihypertensive activity (14). However, the role of Ura in ovarian T/D injury therapy has not been examined yet. Here, we hypothesized that Ura has protective effects against ovarian tissue injury.

Experimental animals and ethical approval
Atatürk University Experimental Animal Ethics

Biochemical examination
10% phosphate buffer solution (PBS) was added to ovarian tissues, and homogenization was performed at 12,000 rpm for 1-2 min on ice (IKA, Germany). The centrifugation process was carried out at +4 °C and 5000 rpm for 30 min to acquire the supernatant, and it was analyzed to determine MDA, TAS, MPO, TOS, and SOD values.
MDA value was assessed via the method described by Ohkawa et al. previously (19) (20). SOD was gauged following reaction with tetrazolium salt to produce formazan dye (21).

Histopathological examination
The rats were sacrificed after euthanasia, and ovarian tissues were incubated in 10% PBS. Then, a routine follow-up process was applied and fixed in paraffin blocks. Sections of 5 μm taken from the blocks were investigated for histopathological changes. Hematoxylin and Eosin staining was performed, and the sections were investigated using a light microscope. They were considered as none (-), mild (+), moderate (++), or severe (+++) (22).

Immunohistochemical (IHC) staining
IHC staining was performed as described in previous studies (23,24). Tissues were kept in a neutral formaldehyde solution for 24 hr and then washed via drinking water. After passing through alcoholxylol follow-up, they were put in paraffin blocks. After deparaffinization of the tissues, they were left for 10 min in 3% H 2 O 2 and washed in phosphate buffer saline (PBS) to prevent peroxidase activity. Then, they were exposed to an antigen retrieval solution at 500w for 10 min. Antigens were removed from tissues by washing in PBS. interleukin-1beta (IL-1β) (Bioss, Cat. No: bs-0812R, Dilution: 1/100) were applied as the primary antibody. 3,3 'diaminobenzidine chromogen was applied, and contrast staining with hematoxylin was done. The samples were analyzed under a light microscope at 20x magnification. Immunopositivity was evaluated as follows: none (−), mild (+), moderate (++), and intense (+++) (25).

Statistical analysis
Statistical analysis was done using SPSS v.20 (IBM, Chicago, USA) software. Nonparametric data were determined using the Kruskal Wallis test. Mann-Whitney U test was used to determine the group that made a difference, and one-way ANOVA was used to evaluate parametric data (26). Tukey's post hoc test was used for multiple comparisons. P<0.05 value was considered significant. All data were expressed as the mean±standard error of the mean (SEM). For IHC staining, all data were analyzed with SPSS statistical software (SPSS for Windows, version 20.0). Data are presented as mean±SEM. For IHC analysis, differences were analyzed with a nonparametric test (Kruskal-Wallis) followed by Mann-Whitney U test (P<0.05) (25).

Results
The results were demonstrated in Tables 1 and 2
T/D and TD+DMSO groups compared with the sham group. In Ura treatment groups, TAS levels increased while TOS and OSI levels diminished. No significant difference was observed in MPO activity between groups. While SOD level decreased in T/D and TD+DMSO groups, it was elevated in Ura treatment groups. MDA levels increased in T/D and TD+DMSO groups while decreasing in Ura administered groups.

Histopathological examination
In the histopathological examination, the ovarian tissues of the sham group had a normal histological appearance ( Figure 1a). In other groups, hemorrhages were observed in the interstitial tissue of the corpus luteum. In T/D ( Figure 1b) and T/D+DMSO (Figure 1c) groups, it was observed that hemorrhage was intense in both the luteal region and stroma. In T/D+Ura 0.5 mg/ kg group (Figure 1d), mild hemorrhage and hyperemia were observed in the stroma. Besides, mild hyperemia was observed in the stroma of the T/D+Ura 5 mg/kg group ( Figure 1e).
When T/D and T/D+DMSO groups were evaluated in terms of hemorrhage, parameters were significantly worse than those of the sham and other treatment groups (Table 3). Significantly fewer histopathological changes were found in T/D+Ura 0.5 mg/kg and T/D+Ura 5 mg/    kg groups compared with T/D and T/D+DMSO groups. There was no statistical difference between sham and T/D+Ura 5 mg/kg groups (Table 3).

IHC examination
There was no immunopositivity for the IHC investigation of IL-1β, TNF-α, and NF-κB in the sham group (Figures 2a, 3a, and 4a). In the T/D group, intensive immunopositivity was found for these parameters. The most intensive immunopositivity for IL-1β was observed in the T/D+DMSO group (Figure 2c). IL-1β immunopositivity declined in both T/D+Ura 0.5 mg/kg ( Figure 2d) and T/D+Ura 5 mg/kg groups (Figure 2e). The IL-1β immunopositivity was significantly higher in the T/D and T/D+DMSO groups than in the sham and other treatment groups (P<0.05, Table 3). However, the IL-1β immunopositivity was significantly lower in the T/D+Ura 5 mg/kg group than in the T/D+Ura 5 mg/ kg group (P<0.05, Table 3). There was no statistical difference between the sham and T/D+Ura 5 mg/kg groups (P>0.05, Table 3).
In the T/D+DMSO group (Figure 3c), TNF-α immunopositivity occurred intensively, but the most intensive immunopositivity was observed in the T/D group (Figure 3b). In treatment groups (Figures 3d  and e), TNF-α immunopositivity decreased. TNF-α immunopositivity of the sham group was significantly lower than the other groups (P<0.05, Table 3). TNF-α immunopositivity increased in the T/D and T/D+DMSO groups (P<0.05, Table 3) and decreased in the T/D+Ura 0.5 mg/kg and T/D+Ura 5 mg/kg groups (P<0.05, Table  3). There was no statistical difference between the T/ D+Ura 0.5 mg/kg and T/D+Ura 5 mg/kg groups (P>0.05, Table 3).
NF-κB immunopositivity was most intense in the T/D+DMSO group (Figure 4c). In T/D (Figure 4b) and T/D+Ura 0.5 mg/kg (Figure 4d) groups, NF-κB immunopositivity was intense, while in T/D+Ura 5 mg/kg group (Figure 4e), NF-κB immunopositivity diminished. Increased NF-kB immunopositivity was observed in T/D and T/D+DMSO groups compared with sham and T/D+Ura 5 mg/kg groups (P<0.05, Table 3). Decreased NF-kB immunopositivity was observed in the T/D+Ura groups, but this decrease only reached a statistical significance in the T/D+Ura 5 mg/kg group (P<0.05, Table 3).
There was no caspase-3 immunopositivity in the sham group (Figure 5a). The most intense caspase-3  Table 3. Histopathological score and immunopositivity evaluation of the experimental groups All data were expressed as mean±SEM. Different superscript letters (a, b, c, d) indicate statistical differences between the groups IL-1β: Interleukin-1 beta; TNF-α: Tumor necrosis factor-alpha; NF-kB: Nuclear factor-kappa B; LC3B: Light chain 3B; DMSO: Dimethyl sulfoxide; Ura: Urapidil; T/D: Torsion detorsion  immunopositivity was seen in the T/D+DMSO group (Figure 5c). In the T/D group (Figure 5b), caspase-3 immunopositivity was less than in the T/D+DMSO group. Besides, caspase-3 immunopositivity was mild in the T/D+Ura 0.5 mg/kg group (Figure 5d), and the lightest immunopositivity occurred in T/D+Ura 5 mg/ kg group (Figure 5e). When the groups were examined, it was shown that the caspase-3 immunopositivity did not occur in the sham group (P<0.05, Table 3). Caspase 3 immunopositivity in the T/D+Ura 5 mg/kg group was significantly lower compared with those in T/D, T/ D+DMSO and T/D+Ura 0.5 mg/kg groups (P<0.05, Table  3). No statistically significant difference was identified between sham and T/D+Ura 5 mg/kg groups in terms of caspase 3 immunopositivity (P>0.05, Table 3). In IHC staining in terms of autophagic cell death, no immune positivity was found in the sham group for LC3B (Figure 6a). In T/D (Figure 6b), T/D+DMSO (Figure  6c), and T/D+Ura 0.5 mg/kg groups (Figure 6d), LC3B immunopositivity was intense, while in T/D+Ura 5 mg/ kg group (Figure 6e), it was mild. When the groups were examined, it was shown that LC3B immunopositivity in the sham group was not found (P<0.05, Table 3). LC3B immunopositivity in the T/D+Ura 5 mg/kg group was significantly lower compared with those in the T/D, T/ D+DMSO, and T/D+Ura 0.5 mg/kg groups (P<0.05, Table  3). No statistically significant difference was identified between the T/D, T/D+DMSO, and T/D+Ura 0.5 mg/ kg groups in terms of LC3B immunopositivity (P>0.05, Table 3).

Discussion
Ovarian torsion is mostly observed during the first 3 decades of life (27). In adolescence, conditions such as excessive tube mobility and overgrowth of the ovaries play a role in the etiology (28). Ischemia duration is important. It has been reported yhat it is possible to return the adnexa to the previous condition within the 18-14 hr following the ischemia (29). Besides, unilateral torsion affects the contralateral ovary in terms of ovulation (30,31). In a previous study, several changes were observed in the contralateral ovaries following ipsilateral ovarian ischemia. This may result from the sympathetic system stimulation by the unilateral ovarian ischemia which lowers the blood flow as in testicular torsion (31).
Ura declines the peripheral vascular resistance acting as an α1-adrenoceptor antagonist (32). By examining   the literature, we can list the healing effects of Ura in this study as follows: First; abnormal catecholamine secretion with complications including myocardial infarction, cardiac failure, and other I/R damage is a major challenge for the treatment management. Ura is a useful and cost-effective choice in the treatment method of patients for example pheochromocytoma perioperative treatment (33). Second; epinephrine and norepinephrine play a role in iron release from ferritin during I/R. This means catecholamines perform a negative activity in the presence of free radicals (34). Catecholamine-iron complexes generate reactive radicals through lipid peroxidases (35). Ura may decline iron ion release from ferritin that may be a potential mechanism of α1-adrenoceptor blocking.
Blood via excessive oxygen amounts during the reperfusion phase leads to neutrophil and ROS formation, damaging the tissue (36). Reperfusion is necessary for oxygen and supplement support but injures tissues via ROS generation and apoptosis (37). High levels of oxygen reach the tissues which leads to excessive ROS formation during the detorsion phase. In our study, TOS, which shows the total value of oxidative stress, and OSI, which shows the balance between antioxidants and oxidants, increased in the study groups in favor of oxidative damage, and these effects reversely changed with Ura treatment. ROS damage DNA and induce lipid peroxidation (38,39). Lipid peroxidation is quite harmful to the cell, which results from free radicals. MDA leads to damage in the cell by enhancing polymerization and cross-linking membrane components (40). In our study, MDA increased due to the damage that occurred in the study groups and decreased with Ura treatment. Antioxidant body mechanisms scavenge ROS and protect tissues. If oxidant mechanisms override the antioxidant systems, this results in increased ROS formation, lipid peroxidation, and tissue injury (41). MPO activity may increase due to I/R injury in ovarian tissues (42). MPO activity indicates neutrophil activation, which is related to ROS generation and cytokine production (43). IL-2, IL-6, IL-1β, and TNF-α are proinflammatory cytokines produced during I/R injury (44,45). IL-1β enhances inflammation and apoptosis rate (46,47). TNF-α is well known as one of the key cytokines mediating inflammatory responses (48). I/R injury enhances inflammation by releasing factors including IL-6, TNF-α, and IL-8. These factors lead to organ damage by playing a role in inflammation (49,50). NF-κB enhances the activation of the various proinflammatory cytokines such as TNF-α, IL-6, COX-2, and IL-1β (51). Reperfusionrelated tissue injury mostly depends on IL-1β and TNF-α (52,53). In this study, IL-1β and TNF-α levels declined due to Ura treatment which supports the decrease of inflammation.
SOD, GPx, and catalase (CAT) prevent the undesirable effects of ROS (54). OSI, the rate of TOS to TAS (55), is a sensitive indicator for oxidative stress assessment (56). ROS generation in the reperfusion stage mainly acts on uncontrolled oxidative stress, and high ROS levels play a role in the inflammatory cascade (57). ROS generation is associated with proapoptotic gene regulation, caspase activation, and apoptosis-related cell death (58). If antioxidants cannot suppress ROS, it results in ovarian damage (59).
Autophagy, cell death, and cell proliferation are related to each other (60). Constantine autophagy modulates apoptosis, and thus, autophagy is a programmed cell death (61). Apoptosis is a self-killing function, and cells with apoptosis are excreted in a controlled manner (62). The caspases also play important roles in the regulation of autophagy, apart from their role in apoptosis. They are activated in response to autophagy (63). Caspase-3 acts as a common effector caspase in all three apoptotic pathways (64). LC3B and caspase-3 expressions demonstrate an increase in various ischemia models (65,66).
In the current study, it was determined that autophagy and apoptosis were triggered during ovarian ischemia. It was also shown that the administration of different doses of Ura suppresses the level of autophagosome marker LC3B and caspase-3. Decreased apoptosis and autophagy following ischemic damage in Ura groups are associated with autophagosome marker LC3B.

Conclusion
Our study showed that Ura can elevate SOD and TAS activity, and reduce the TOS level significantly in ovarian tissue injury. Histopathological analysis showed that Ura reduced inflammatory cytokines, apoptosis, and autophagy with rising doses. In conclusion, Ura has a protective effect against ovarian T/D injury in rats.